Human hematopoietic stem cells (HSC) transplantation is currently being used as regenerative medicine for the treatment of congenital deficiencies and malignant diseases as well as cancers and other disorders of the blood and immune systems. However, despite all the enthusiasm surrounding HSC biology and therapeutics, the potential of HSC-based therapies has yet to be fully realized. A major roadblock of broader use of the adult stem cells is the limited number of HSC per harvest for therapeutic benefit and their poorly understood expansion and differentiation behavior in response to proliferative stimuli. In vitro expansion of HSC remains a major challenge for wide applications of HSC transplantation for patients. Our studies show that p18, a member of the cyclin-dependent kinase (CDK) inhibitors (CKI), is a potent negative regulator of HSC self- renewal. Thus, we hypothesize that p18 is a unique drug target, and that small molecules capable of blocking p18 function and interfering with p18/CDK6 interactions are likely to be potent drugs for activating HSC self- renewal. Our objective is to screen/identify p18 inhibitors that act by disrupting p18/CDK6 interactions, thus activating HSC self-renewal and increasing the quantity of active stem cells, and to use them as chemical probes for mechanism studies of HSC self-renewal. The feasibility of the proposed innovative research is supported by the proof-of-principle pilot data obtained by well-established research teams that have complementary expertise for the proposed research. Considering the limited throughput capacity of the current HSC bone-marrow culture protocol, we propose first to use our established in silico screening approach for initial screening to generate p18-focused lead sublibraries (Aim 1). We also apply NMR assays to screen/validate and characterize the p18 hits and their binding interactions with the protein in order to generate p18-active subsets (Aim 2A). Also, the small subsets of validated p18-targeting compounds will then be confirmed by extensive HSC functional assays (Aim 2B). Through these, the compounds that are capable of increasing the number of active stem cells in the bone-marrow culture will be identified as leads. The discovered leads are then used as specific chemical probes for studies of p18/CDK6 interactions and signaling mechanisms of the G1-phase of the cell cycle. As a future plan, the identified leads will be further optimized by chemistry modification and SAR medicinal chemistry studies to improve the potency and cell toxicity. Our long- term goal is to identify/design CKI p18-specific small molecule effectors that can either maintain/stimulate self- renewal of hematopoietic stem cells in a predictable manner, and ultimately to develop new drugs for HSC therapies. Achieving this goal will have a significant impact on stem cell drug research development in general.